Study On Screening Of Pine Needles Degrading Strains And Its Degradation Process

Pine trees,as one of the main plants in the pine green environment,are widely grown in most parts of China.Pine needles,which are the needles of pinaceae plants genus,have an abundant resources.As a cheap and sustainable renewable natural resource,pine needles have great research value.The discarded needles are rationally used as a soil conditioner,which can effectively improve the soil degradation,soil organic content and soil biodiversity.Therefore,the development and utilization of pine needles resource is vital to the improvement of soil ecological environment.In this research,the humus samples were collected in the Great Khingan Pines area,screened by pine needle powder domestication selection medium as the sole carbon source,and dyed by Congo red staining.The larger ratio of hydrolysis zone diameter and colony diameter and faster growing strains were selected,which nine strains were ZJ-1,ZJ-2,ZJ-5,ZJ-8,ZJ-11,ZJ-13,ZJ-15,ZJ-16 and ZJ-19.Then three efficient cellulase production and degradation of pine needles strains respectively ZJ-8,ZJ-13 and ZJ-19 were selected by affination of the fermentation liquid cellulase enzyme activity assay.The molecular identification results showed that ZJ-8 strains was Aspergillus awamori,ZJ-13 was Penicillium and ZJ-19 was Hypocrea koningii.These three strains were cultivated with the laboratory-deposited strain 38(strain 38 is Bacillus subtilis).By measuring enzyme activity,the enzyme activities of most strain composition were higher than the strains which were cultured alone.The optimal combination of flora with the highest enzyme activity was the combination of 38,ZJ-8 and ZJ-13,which was selected for the further research.The result of preliminary study of the optimal combination solid-state fermentation conditions showed that sodium nitrate as nitrogen source and initial pH6.0 with alcohol 400 as the surfactant polyethylene leaded the optimal enzyme production.The measured CMC enzyme activity was 98.03IU,FPA enzyme activity was 50.84IU and microcrystalline cellulose enzyme activity was 140.06IU.The FTIR was used to study the biodegradation of pine needles which can be a more detailed view of the dynamic changes of the main components' chemical structure of pine needles.Pine needles contain carbohydrates,lignin organic silicide and other ingredients,which can become carbonates,silicates and polysaccharides in the degradation.Carboxylic acid salt can be formed with the carboxyl in the fermentation process and carbonate can be formed with several inorganic cations.Meanwhile,scanning electron microscopy is used to study dynamic process of pine needles degradation during period of 70 days.The results showed that during initial biodegradation mycelium invaded into the interior through the vent and the end portion of pine needles and reproducted in the lumen of parenchyma cells,then parenchyma cells began to decompose,waxy-silicide layer became significantly thinner.When thallus was mass-producted,the vascular tissue and parts of the thick tissue began to degrade.At the end of degradation test,the complete structure of pine needles was severely damaged,all parenchyma was degraded,waxy-silicide layer basically was completely degraded only leaving some the highly lignified epidermal tissues and sclerenchyma.